Genetic analysis of apoptotic cell clearance in flies

果蝇凋亡细胞清除的遗传分析

• 批准号: 7107264
• 负责人: KRISTIN WHITE
• 金额: $ 30.76万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7107264
• 关键词: Drosophilidae; RNA interference; apoptosis; arthropod genetics; biological signal transduction; cell surface receptors; differential display technique; gene expression; gene expression profiling; phagocytosis; phenotype; phosphatidylserines; protein structure function; tissue /cell culture

DESCRIPTION (provided by applicant): Apoptotic cells are rapidly cleared by phagocytosis in vivo. Defects in apoptotic cell phagocytosis have been implicated in several human pathologies such as Systemic Lupus Erythematosus. In the absence of engulfment, the remaining tissue may be damaged by exposure to the contents of the dying cells. In Drosophila, apoptotic corpses are rapidly engulfed by circulating phagocytic cells. We have shown that the croquemort gene is required for the efficient engulfment of apoptotic cells in the embryo. We propose to identify and characterize other genes involved in engulfment, using a combination of Drosophila genetics, tissue culture assays and differential expression screens. A screen of genomic deletions identified a number of regions required for efficient engulfment of apoptotic cells in the embryo. One of the genomic regions identified in our screen contains the Drosophila homologue of the phosphatidylserine receptor. This receptor has been implicated in the phagocytosis of apoptotic corpses in mammalian tissue culture assays. Although we do not detect a major role for this receptor in engulfment in the Drosophila embryo, we do find that it functions to inhibit apoptosis. We will use this phenotype to screen for signaling pathways activated by the receptor. In addition, we will further explore the requirement for this receptor in engulfment. These studies will provide insight into the general function of the protein. We have also identified other genomic regions that contain genes required for engulfment. We will distinguish the responsible genes using genetic and RNA interference techniques. A tissue culture based apoptotic cell engulfment assay has been developed to facilitate RNAi techniques. This will allow us to examine the role of genes required in both the phagocyte and in the apoptotic target cell. In our initial studies, we found that croquemort levels were upregulated in the presence of apoptotic cells. To identify other genes in the phagocyte that show changes in expression on exposure to apoptotic cells, we have initiated a differential expression screen. Genes that show expression changes in response to the presence of apoptotic cells will be tested for a role in engulfment in tissue culture and in vivo. This combination of strategies will allow us to identify and characterize many genes involved in apoptotic cell engulfment, and to understand how they function in this important process.

描述（由申请人提供）：体内凋亡细胞通过吞噬作用迅速清除。凋亡细胞吞噬功能的缺陷与一些人类疾病有关，如系统性红斑狼疮。在没有吞噬的情况下，剩余的组织可能因暴露于死亡细胞的内容物而受损。在果蝇中，凋亡的尸体被循环的吞噬细胞迅速吞噬。我们已经证明，croquemort基因是有效吞噬胚胎中凋亡细胞所必需的。我们建议使用果蝇遗传学、组织培养试验和差异表达筛选相结合的方法来鉴定和表征参与吞噬的其他基因。基因组缺失的筛选确定了胚胎中有效吞噬凋亡细胞所需的一些区域。在我们的筛选中发现的一个基因组区域包含果蝇的磷脂酰丝氨酸受体同源物。在哺乳动物组织培养实验中，这种受体与凋亡尸体的吞噬作用有关。虽然我们没有发现这种受体在果蝇胚胎的吞噬中起主要作用，但我们确实发现它具有抑制细胞凋亡的功能。我们将使用这种表型来筛选由受体激活的信号通路。此外，我们将进一步探讨吞噬对该受体的要求。这些研究将对这种蛋白质的一般功能提供深入的了解。我们还发现了其他包含吞噬所需基因的基因组区域。我们将使用遗传和RNA干扰技术区分负责基因。一种基于组织培养的凋亡细胞吞噬实验已经被开发出来，以促进RNAi技术。这将使我们能够检查在吞噬细胞和凋亡靶细胞中所需的基因的作用。在我们最初的研究中，我们发现在凋亡细胞存在的情况下，croquemort水平上调。为了鉴定吞噬细胞中在暴露于凋亡细胞时表现出表达变化的其他基因，我们启动了差异表达筛选。在组织培养和体内实验中，对凋亡细胞的表达变化的基因将进行测试，以确定其在吞噬中的作用。这种策略的结合将使我们能够识别和表征许多参与凋亡细胞吞噬的基因，并了解它们在这一重要过程中的作用。